scholarly journals CYTOCHEMICAL LOCALIZATION OF 5'-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER

1972 ◽  
Vol 52 (3) ◽  
pp. 542-558 ◽  
Author(s):  
Christopher C. Widnell
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Ethylenediaminetetraacetic Acid ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Cytochemical Localization ◽  
Low Concentrations ◽  
Microsomal Membranes ◽  
Microsome Fraction

A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO3)2 and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb2+, or Pb2+ and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb2+, and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb2+ and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb2+ and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.

scholarly journals Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes

1991 ◽  
Vol 280 (1) ◽  
pp. 171-178 ◽  
Author(s):  
L M Traub ◽  
E Shai ◽  
R Sagi-Eisenberg
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Trypsin Treatment ◽  
Putative Receptor ◽  
Microsomal Membranes ◽  
Direct Binding ◽  
Triton X ◽  
Binding Sequence

p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.

scholarly journals Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion

1997 ◽  
Vol 323 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Mark R. OWEN ◽  
Clark C. CORSTORPHINE ◽  
Victor A. ZAMMIT
Keyword(s):  
Rat Liver ◽  
Low Density Lipoprotein ◽  
Mode Of Delivery ◽  
Liver Microsomes ◽  
Density Lipoprotein ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
Rat Liver Microsomes ◽  
Microsomal Membranes ◽  
Latent Activity

The possibility that triacylglycerol (TAG) synthesis occurs on both aspects of the endoplasmic-reticular membrane during the process of incorporation of TAG into secreted very-low-density lipoprotein (VLDL) [Zammit (1996) Biochem. J. 314, 1-14] was investigated by measuring the latency of diacylglycerol acyltransferase (DGAT) in microsomal fractions obtained from rat liver homogenates. Permeabilization of microsomes with taurocholate resulted in the doubling of the activity, indicating that DGAT activities of approximately equal magnitude occur on either aspect of the microsomal membrane. The taurocholate concentrations required for exposure of the latent activity of DGAT were identical with those that resulted in the exposure of marker enzymes for the lumen of the endoplasmic reticulum. Fractionation of the microsomes into smooth and rough populations indicated that the distribution of overt and latent DGAT activities was the same throughout. The possibility that taurocholate effects may result from non-specific activation of the overt enzyme was excluded by employing the channel-forming peptide alamethicin to effect permeabilization, and by varying the mode of delivery of diacylglycerol substrate to the microsomal membranes. Permeabilization using alamethicin gave a slightly higher latent/overt ratio for DGAT. The possible roles of overt and latent DGAT activities in the synthesis and secretion of TAG by the liver are discussed.

scholarly journals Effects of CoA and acyl-CoAs on GTP-dependent Ca2+ release and vesicle fusion in rat liver microsomal vesicles

1993 ◽  
Vol 289 (2) ◽  
pp. 561-567 ◽  
Author(s):  
J G Comerford ◽  
A P Dawson
Keyword(s):  
Chain Length ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Carbon Chain ◽  
Vesicle Fusion ◽  
Carbon Chain Length ◽  
Rat Liver Microsomes ◽  
Low Concentrations ◽  
Ic50 Values ◽  
Pumping Activity

(1) CoA (IC50 23 microM) and acyl-CoAs (IC50 values 15-18 microM) inhibit GTP-dependent vesicle fusion in rat liver microsomal vesicles. Acyl-CoAs of carbon chain length C8 and C20 are much less effective than acyl-CoAs of carbon chain length C14-C18. The effect of CoA is mimicked by dephospho-CoA, but not by desulpho-CoA. High acyl-CoA concentrations (50 microM) appear to favour formation of small vesicles (budding), while 50 microM CoA does not. (2) Low concentrations of CoA (EC50 2 microM) and palmitoyl-CoA (10 microM) cause re-accumulation of Ca2+ released in response to GTP. This re-accumulation is into an Ins(1,4,5)P3-sensitive compartment. By investigation of the effects of CoA and palmitoyl-CoA on the thapsigargin-induced passive leak rate of Ca2+, and on the latency of the mannose-6-phosphatase of the vesicles, we conclude that CoA and palmitoyl-CoA cause decreased vesicle permeability rather than stimulation of Ca2+ pumping activity. (3) It is suggested that GTP-induced membrane fusion in rat liver microsomes involves an as yet uncharacterized acylation-deacylation reaction which is required to produce complete vesicle sealing.

scholarly journals A Biochemical and Morphological Study of Rat Liver Microsomes

1960 ◽  
Vol 7 (3) ◽  
pp. 547-557 ◽  
Author(s):  
Y. Moulé ◽  
C. Rouiller ◽  
J. Chauveau
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Sucrose Solution ◽  
Liver Microsomes ◽  
Liver Homogenate ◽  
Sodium Deoxycholate ◽  
Point Of View ◽  
Rat Liver Microsomes ◽  
Experimental Conditions ◽  
Microsome Fraction

Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point of view. 1. Under these experimental conditions, the "total microsome" fraction was obtained by centrifuging the cytoplasmic extract free of nuclei and mitochondria, for 3 hours at 145,000 g. Morphologically, the total microsomes consist mainly of "rough-surfaced membranes" and "smooth" ones. 2. The total microsomes have been divided into 2 subfractions so that the 1st microsomal fraction contains the "rough" vesicles (2 hours centrifugation at 40,000 g) while the 2nd microsomal fraction consists essentially of smooth vesicles, free particles, and ferritin (centrifugation of the supernatant at 145,000 g for 3 hours). 3. By the action of 0.4 per cent sodium deoxycholate in 0.88 M sucrose, it was possible to obtain a pellet for each of the 2 fractions which consisted of dense particles, rich in RNA, poor in lipids, and which represented about 50 to 60 percent of the RNA and 10 to 15 per cent of the proteins. The results have been discussed taking into consideration the hypothesis of the presence of RNA in the membranes of microsomal vesicles.

scholarly journals Degradation of Stearoyl-Coenzyme A Desaturase: Endoproteolytic Cleavage by an Integral Membrane Protease

1998 ◽  
Vol 9 (12) ◽  
pp. 3445-3453 ◽  
Author(s):  
F. Scott Heinemann ◽  
Juris Ozols
Keyword(s):  
Rat Liver ◽  
Coenzyme A ◽  
Liver Microsomes ◽  
Integral Membrane Protein ◽  
Rat Liver Microsomes ◽  
Selective Degradation ◽  
Endoproteolytic Cleavage ◽  
Microsomal Membranes ◽  
Normal Rat

Stearoyl-coenzyme A desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a prototype for selective degradation of resident proteins of the endoplasmic reticulum. Using detergent-solubilized, desaturase-induced rat liver microsomes we have characterized a protease that degrades SCD. Degradation of SCD in vitro is highly selective, has a half-life of 3–4 h, and generates a 20-kDa C-terminal fragment of SCD. The N terminus of the 20-kDa fragment was identified as Phe177. The cleavage site occurs in a conserved 12-residue hydrophobic segment of SCD flanked by clusters of basic residues. The SCD protease remains associated with microsomal membranes after peripheral and lumenal proteins have been selectively removed. SCD protease is present in normal rat liver microsomes and cleaves purified SCD. We conclude that rapid turnover of SCD involves a constitutive microsomal protease with properties of an integral membrane protein.

Effect of nucleotides on UDP-N-acetylglucosamine pyrophosphatase and N-acetylglucosaminyltransferase activities in microsomal membranes

1979 ◽  
Vol 57 (6) ◽  
pp. 557-565 ◽  
Author(s):  
Sailen Mookerjea
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Rat Liver Microsomes ◽  
Glycoprotein Synthesis ◽  
Microsomal Membranes ◽  
Pyrophosphatase Activity ◽  
Triton X ◽  
Hydrolysis Of

Rat liver microsomes solubilized by incubating with lysolecithin or Triton X-100 showed very active UDP-N-acetylglucosamine pyrophosphatase activity leading to the hydrolysis of the substrate into N-acetylglucosamine-1-P and N-acetylglucosamine. ATP, GTP, CDPcholine, and CDPglucose exerted a considerable inhibitory effect on the solubilized membrane pyrophosphatase activity. CDPcholine and CDPglucose, in addition, appeared to stimulate the transfer of N-acetylglucosamine into endogenous and exogenous acceptor proteins. Evidence is also presented of an inhibitory effect of ATP (and to some extent GTP) on N-acetylglucosaminyltransferase activity. This inhibitory effect of ATP and GTP became clearly evident when the pyrophosphatase activity in the membranes was virtually eliminated in the presence of CDPcholine and CDPglucose. The effect of ATP and GTP on the solubilized membrane enzymes indicated that the inhibition of pyrophosphatase activity alone did not determine the rate of transfer of sugar to protein. The results also suggested that the UDP-N-acetylglucosamine pyrophosphatase and N-acetyiglucosaminyltransferase activities were controlled independently and the effect of each nucleotide on these enzymes should, therefore, be carefully evaluated to understand its role in glycopolymer biosynthesis. Also, a possible role of choline and its derivatives in glycoprotein synthesis is discussed.

scholarly journals Ruthenium red-insensitive calcium transport in ascites-sarcoma 180/TG cells

1981 ◽  
Vol 200 (2) ◽  
pp. 343-348 ◽  
Author(s):  
F L Bygrave ◽  
T A Anderson
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Initial Rate ◽  
Liver Microsomes ◽  
Ruthenium Red ◽  
Rat Liver Microsomes ◽  
Sarcoma 180 ◽  
Low Concentrations ◽  
Rat Liver Cells ◽  
Mouse Ascites

1. Ruthenium Red-insensitive Ca2+ transport in the mouse ascites sarcoma 180/TG is enriched in a ‘heavy’ microsomal fraction (microsomes) sedimented at 35 000 g for 20 min. The subcellular distribution of this Ca2+ transport differed from that of Ruthenium Red-sensitive Ca2+ transport and (Na+ + K+)-dependent ATPase activity, but was similar to that of glucose 6-phosphatase. 2. The affinity of this transport system for ‘free’ Ca2+ is high (Km approx. 6 microM) and that for MgATP somewhat lower (Km approx. 100 microM). Ca2+ transport by the tumour microsomes, by contrast with that by liver microsomes, was greatly stimulated by low concentrations of P1. 3. Although incubation of intact ascites cells with glucagon led to an increase in intracellular cyclic AMP, no stable increase in the initial rate of Ca2+ transport in the subsequently isolated ‘heavy’ microsomes could be detected as in similar experiments carried out previously with rat liver cells. Reconstitution experiments suggest that a deficiency exists in the tumour microsomal membrane such that an action of glucagon that is normally present in rat liver microsomes is not evoked.

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